Hepatitis Delta Antigen Mediates the Nuclear Import of Hepatitis Delta Virus RNA

Hepatitis delta virus (HDV) RNA replicates in the nuclei of virus-infected cells. The mechanism of nuclear import of HDV RNA is so far unknown. Using a fluorescein-labeled HDV RNA introduced into partially permeabilized HeLa cells, we found that HDV RNA accumulated only in the cytoplasm. However, in the presence of hepatitis delta antigen (HDAg), which is the only protein encoded by HDV RNA, the HDV RNA was translocated into the nucleus, suggesting that nuclear import of HDV RNA is mediated by HDAg. Deletion of the nuclear localization signal (NLS) or RNA-binding motifs of HDAg resulted in the failure of nuclear import of HDV RNA, indicating that both the NLS and an RNA-binding motif of HDAg are required for the RNA-transporting activity of HDAg. Surprisingly, any one of the three previously identified RNA-binding motifs was sufficient to confer the RNA-transporting activity. We have further shown that HDAg, via its NLS, interacts with karyopherin α2 in vitro, suggesting that nuclear import of the HDAg-HDV RNA complex is mediated by the karyopherin α2β heterodimer. The nuclear import of HDV RNA may be the first biological function of HDAg in the HDV life cycle.     

Genotype-Specific Complementation of Hepatitis Delta Virus RNA Replication by Hepatitis Delta Antigen

Characterizations of genetic variations among hepatitis delta virus (HDV) isolates have focused principally on phylogenetic analysis of sequences, which vary by 30 to 40% among three genotypes and about 10 to 15% among isolates of the same genotype. The significance of the sequence differences has been unclear but could be responsible for pathogenic variations associated with the different genotypes. Studies of the mechanisms of HDV replication have been limited to cDNA clones from HDV genotype I, which is the most common. To perform a comparative analysis of HDV RNA replication in genotypes I and III, we have obtained a full-length cDNA clone from an HDV genotype III isolate. In transfected Huh-7 cells, the functional roles of the two forms of the viral protein, hepatitis delta antigen (HDAg), in HDV RNA replication are similar for both genotypes I and III; the short form is required for RNA replication, while the long form inhibits replication. For both genotypes, HDAg was able to support replication of RNAs of the same genotype that were mutated so as to be defective for HDAg production. Surprisingly, however, neither genotype I nor genotype III HDAg was able to support replication of such mutated RNAs of the other genotype. The inability of genotype III HDAg to support replication of genotype I RNA could have been due to a weak interaction between the RNA and HDAg. The clear genotype-specific activity of HDAg in supporting HDV RNA replication confirms the original categorization of HDV sequences in three genotypes and further suggests that these should be referred to as types (i.e., HDV-I and HDV-III) rather than genotypes.     

庚型肝炎病毒全长基因在体外的表达

利用第二军医大学微生物学教研室克隆的庚型肝炎病毒 (HGV)全长基因组 (HGVqz) ,构建由不同启动子调控的HGV表达载体 ,将 2种表达载体及HGV全长基因片段进行体外表达 ,探讨此HGV全长基因克隆的功能。利用脂质体将HGV全长基因cDNA以及表达载体转入Changliver或NIH 3T3细胞进行瞬时表达 ,分别提取转染72h后转染细胞的RNA及蛋白质进行RT PCR及Westernblotting ,以检测HGV基因的表达。RT PCR及Westernblotting结果表明 ,HGV全长基因cDNA以及 2种表达载体均可以在体外培养的细胞内表达 ,其表达产物为HGV前体蛋白 ,分子量约为 310ku。第二军医大学微生物学教研室克隆的HGV全长基因具有正确的开读框架和可表达性 ,能表达HGV前体蛋白 ,但在体外培养细胞内不能完成前体蛋白的剪切     

cis-Acting Signals on the RNAs of Hepatitis Delta Virus

The replication of the genomic RNA of hepatitis delta virus involves RNA-directed RNA synthesis and various forms of RNA processing, including ribozyme cleavage, polyadenylation, and RNA editing. The purpose of this article is to evaluate the evidence concerning eight sequence elements that may contribute to the direction of these different events.     

Arginine-Rich Motifs Are Not Required for Hepatitis Delta Virus RNA Binding Activity of the Hepatitis Delta Antigen

Hepatitis delta virus (HDV) replication and packaging require interactions between the unbranched rodlike structure of HDV RNA and hepatitis delta antigen (HDAg), a basic, disordered, oligomeric protein. The tendency of the protein to bind nonspecifically to nucleic acids has impeded analysis of HDV RNA protein complexes and conclusive determination of the regions of HDAg involved in RNA binding. The most widely cited model suggests that RNA binding involves two proposed arginine-rich motifs (ARMs I and II) in the middle of HDAg. However, other studies have questioned the roles of the ARMs. Here, binding activity was analyzed in vitro using HDAg-160, a C-terminal truncation that binds with high affinity and specificity to HDV RNA segments in vitro. Mutation of the core arginines of ARM I or ARM II in HDAg-160 did not diminish binding to HDV unbranched rodlike RNA. These same mutations did not abolish the ability of full-length HDAg to inhibit HDV RNA editing in cells, an activity that involves RNA binding. Moreover, only the N-terminal region of the protein, which does not contain the ARMs, was cross-linked to a bound HDV RNA segment in vitro. These results indicate that the amino-terminal region of HDAg is in close contact with the RNA and that the proposed ARMs are not required for binding HDV RNA. Binding was not reduced by mutation of additional clusters of basic amino acids. This result is consistent with an RNA-protein complex that is formed via numerous contacts between the RNA and each HDAg monomer.     

A Novel Diagnostic Target in the Hepatitis C Virus Genome

Background

Detection and quantification of hepatitis C virus (HCV) RNA is integral to diagnostic and therapeutic regimens. All molecular assays target the viral 5′-noncoding region (5′-NCR), and all show genotype-dependent variation of sensitivities and viral load results. Non-western HCV genotypes have been under-represented in evaluation studies. An alternative diagnostic target region within the HCV genome could facilitate a new generation of assays.

Methods and Findings

In this study we determined by de novo sequencing that the 3′-X-tail element, characterized significantly later than the rest of the genome, is highly conserved across genotypes. To prove its clinical utility as a molecular diagnostic target, a prototype qualitative and quantitative test was developed and evaluated multicentrically on a large and complete panel of 725 clinical plasma samples, covering HCV genotypes 1–6, from four continents (Germany, UK, Brazil, South Africa, Singapore). To our knowledge, this is the most diversified and comprehensive panel of clinical and genotype specimens used in HCV nucleic acid testing (NAT) validation to date. The lower limit of detection (LOD) was 18.4 IU/ml (95% confidence interval, 15.3–24.1 IU/ml), suggesting applicability in donor blood screening. The upper LOD exceeded 10⁻⁹ IU/ml, facilitating viral load monitoring within a wide dynamic range. In 598 genotyped samples, quantified by Bayer VERSANT 3.0 branched DNA (bDNA), X-tail-based viral loads were highly concordant with bDNA for all genotypes. Correlation coefficients between bDNA and X-tail NAT, for genotypes 1–6, were: 0.92, 0.85, 0.95, 0.91, 0.95, and 0.96, respectively; X-tail-based viral loads deviated by more than 0.5 log10 from 5′-NCR-based viral loads in only 12% of samples (maximum deviation, 0.85 log10). The successful introduction of X-tail NAT in a Brazilian laboratory confirmed the practical stability and robustness of the X-tail-based protocol. The assay was implemented at low reaction costs (US$8.70 per sample), short turnover times (2.5 h for up to 96 samples), and without technical difficulties.

Conclusion

This study indicates a way to fundamentally improve HCV viral load monitoring and infection screening. Our prototype assay can serve as a template for a new generation of viral load assays. Additionally, to our knowledge this study provides the first open protocol to permit industry-grade HCV detection and quantification in resource-limited settings.     

Pseudoplusia includens Densovirus Genome Organization and Expression Strategy

The genome of a densovirus of a major phytophagous pest, Pseudoplusia includens, was analyzed. It contained 5,990 nucleotides (nt) and included inverted terminal repeats of 540 nt with terminal Y-shaped hairpins of 120 nt. Its DNA sequence and ambisense organization with 4 typical open reading frames demonstrated that it belonged to the genus Densovirus in the subfamily Densovirinae of the family Parvoviridae.     

Hormone-activated Expression of the C-type RNA Tumour Virus Genome

THE concept of vertical transmission of specific viral information, particularly that possibly associated with the induction of malignancy in mice has been postulated. Moreover, it has been hypothesized that this genetic information may be expressed either in the form of whole virus in certain selected laboratory animal strains or as the operon involved in regulating cellular replication (oncogene)¹. To detect this proposed genetically transmitted message, one uses a group specific antisera against the C-type RNA tumour viruses having as one of its components, gs-3, first described by Gerring et al.². A similar group specific antigen has subsequently been reported by Schafer³, Gilden⁴ and Sarma et al.⁵ and designated “interspec”, meaning that the antigen is common to the internal components of the C-type RNA virion of several mammalian species.     

Complete Nucleotide Sequence and Genome Organization of Soybean Crinkle Leaf Virus

The genomic DNA of soybean crinkle leaf virus (SCLV) from Thailand has been sequenced. The single circular DNA molecule comprises 2737 nucleotides, and contains eight open reading frames each capable of encoding a protein with a molecular weight greater than 10 kDa. A 39‐base potential stem‐loop forming region occurs in the intergenic region (IR) that also includes the conserved nonanucleotide sequence TAATATTAC. The iterative sequence (TCAATCGGTGT), which is specific to SCLV, is also found in the IR. SCLV is most closely related (90% identity) to the monopartite geminivirus ageratum yellow vein virus. As the two viruses differ in host range, and the iterative sequence is specific to SLCV, the virus is a distinct monopartite geminivirus of soybean.     

Clathrin-Mediated Post-Golgi Membrane Trafficking in the Morphogenesis of Hepatitis Delta Virus

Clathrin is involved in the endocytosis and exocytosis of cellular proteins and the process of virus infection. We have previously demonstrated that large hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis delta virus (HDV) are not clear. In this study, we found that clathrin heavy chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a single amino acid substitution at the clathrin box retained nuclear export activity but failed to interact with CHC and to assemble into virus-like particles. Downregulation of CHC function by a dominant-negative mutant or by short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion of hepatitis B virus subviral particles. In addition, the coexistence of a cell-permeable peptide derived from the C terminus of HDAg-L significantly interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and CHC were found to colocalize with the trans-Golgi network and were highly enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which assembles less efficiently than genotype I HDV does, has a putative clathrin box in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of the various HDV genotypes correlates well with the CHC-binding activity of their HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin box and the nuclear export signal at the C terminus of HDAg-L are potential new molecular targets for HDV therapy.Pathogens often take advantage of intracellular pathways involved in the trafficking of cellular macromolecules in order to carry out their life cycle, which consists of virus entry, translation, genome replication, assembly, and release. The clathrin-mediated endocytic route is a pathway commonly used for virus entry (29). Following clathrin-mediated endocytosis, incoming viruses are transported together with their receptors from the plasma membrane into early and late endosomes. Several links between clathrin adaptor complexes and viral biogenesis, including those of influenza virus (37), reovirus (13), and vesicular stomatitis virus (33), have been demonstrated.Clathrin and its adaptor proteins (APs), which constitute the major components of clathrin-coated vesicles (CCVs), are often the carriers of proteins and lipids that are transported from the trans-Golgi network (TGN) to the endosome (20, 35). Clathrin-mediated exocytosis has been found to participate in viral multiplication. The envelope protein of vesicular stomatitis virus, glycoprotein 1, recruits clathrin adaptor complex adaptor protein 1 (AP1) onto Golgi membranes and possibly leaves the TGN in CCVs for subsequent transport to endosomes (1). It is also known that interaction of AP1 with the matrix domain of human immunodeficiency virus type 1 Gag protein promotes viral release (5). In addition, Vpu inhibits the endosomal accumulation of the human immunodeficiency virus type 1 structural proteins Env and Gag, which is known to enhance viral assembly and release at the plasma membrane (39). Furthermore, large hepatitis delta antigen (HDAg-L) encoded by the hepatitis delta virus (HDV) has recently been identified as a novel clathrin adaptor-like protein (18). HDAg-L specifically interacts with clathrin heavy chain (CHC) at the TGN and inhibits clathrin-mediated protein transport. However, the role of CHC in the life cycle of HDV remains unclear.HDV is a highly pathogenic virus. The virion is coated with the envelope proteins of hepatitis B virus (HBV), the hepatitis B virus surface antigens (HBsAgs) (24). Superinfection or coinfection with HBV may result in fulminant hepatitis and progressive chronic liver cirrhosis (3, 36). The small HDAg (HDAg-S) lacks the unique C-terminal 19-amino-acid sequence of HDAg-L (6, 41, 43) and functions as a transactivator of HDV genome replication in the nucleus (23, 24). Both HDAg-S and HDAg-L possess nuclear localization signals (NLSs) spanning amino acid residues 35 to 88 and are mainly localized in the nuclei of transfected cells in the absence of HBsAg (7, 8). However, HDAg-L has been demonstrated to be a nucleocytoplasmic shuttling protein with a nuclear export signal (NES) at its unique C terminus, and this is important for HDV assembly (27). In the presence of HBsAg, HDAg-L relocalizes to the cytoplasm (29). In addition, a NES-interacting protein of HDAg-L, NESI, has been identified to be essential for the HDAg-L-mediated nuclear export of HDV RNA (42). Furthermore, the proline-rich motif within the unique 19-amino-acid extension together with isoprenylation of the CXXX motif (15) are essential for HDAg-L to form delta virus-like particles (VLPs) with HBsAg (19, 22). Taken together, these results imply that an intracellular association between HDAg-L and HBsAg in the cytoplasm is the driving force of HDV assembly. The interaction of HDAg-L with HBsAg facilitates the assembly and secretion of HDV particles. Nevertheless, the cellular proteins and pathways involved in the transport, packaging, and secretion of HDV are poorly understood.In this study, the involvement of clathrin-mediated trafficking in the propagation of HDV is biochemically characterized. Downregulation of functional CHC significantly reduced the efficiency of the CCV-mediated HDV assembly. However, CHC is not essential for the assembly of HBV subviral particles (SVPs). These results indicate that, although HBV and HDV share common surface antigens, different mechanisms are involved in their viral assembly and release. In addition, the assembly efficiency of the various HDV genotypes correlates well with the ability of HDAg-L to interact with CHC. This may reflect the fact that there is lower pathogenicity among patients infected with HDV genotype II than among those infected with genotype I.     

Electrostatic Regulation of Genome Packaging in Human Hepatitis B Virus

Hepatitis B virus (HBV) is a contagious human pathogen causing liver diseases such as cirrhosis and hepatocellular carcinoma. An essential step during HBV replication is packaging of a pregenomic (pg) RNA within the capsid of core antigens (HBcAgs) that each contains a flexible C-terminal tail rich in arginine residues. Mutagenesis experiments suggest that pgRNA encapsidation hinges on its strong electrostatic interaction with oppositely charged C-terminal tails of the HBcAgs, and that the net charge of the capsid and C-terminal tails determines the genome size and nucleocapsid stability. Here, we elucidate the biophysical basis for electrostatic regulation of pgRNA packaging in HBV by using a coarse-grained molecular model that explicitly accounts for all nonspecific interactions among key components within the nucleocapsid. We find that for mutants with variant C-terminal length, an optimal genome size minimizes an appropriately defined thermodynamic free energy. The thermodynamic driving force of RNA packaging arises from a combination of electrostatic interactions and molecular excluded-volume effects. The theoretical predictions of the RNA length and nucleocapsid internal structure are in good agreement with available experiments for the wild-type HBV and mutants with truncated HBcAg C-termini.     

Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.     

Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication.